Method and apparatus for electrically assisted topical delivery of agents for cosmetic applications

ABSTRACT

The present invention is based on the development of a method for cosmetic delivery of L-ascorbic acid-containing compositions to the layer of the skin wherein collagen formation takes place to enhance production of collagen and thereby combat some of the effects of aging and oxy-radical damage on skin. Sufficient electric pulses applied to a region of skin surface temporarily create new pathways through the lipid skin barrier, thereby delivering an L-ascorbic acid-containing composition through the lipid skin barrier and enhancing production of collagen in the region of the skin so treated. A handheld electrical pulser apparatus, optionally table-top adaptable, and methods of use for cosmetic treatment of degenerative skin conditions are provided by the invention.

RELATED APPLICATION

This application is a continuation of application Ser. No. 09/352,818,filed Jul. 13, 1999, now U.S. Pat. No. 6,302,874.

This application relies for priority under 35 U.S.C. §119(e)(1) onprovisional application Ser. No. 60/092,541, filed Jul. 13, 1998.

FIELD OF THE INVENTION

The present invention generally relates to methods for enhancing theeffectiveness of cosmetic pharmaceuticals used to improve the appearanceof skin. In particular, the present invention relates to use ofelectroporation-mediated topical delivery of agents, such as Vitamin C.

BACKGROUND OF THE INVENTION

The main factors causing skin aging are natural processes (such asaging), lifestyle factors (such as smoking), and environmental stressors(such as UV radiation, chemical pollutants, etc.). It is now medicallyrecognized that many of these factors damage skin through production ofoxy-radical damage. Superoxide and the subsequently generated hydrogenperoxide and hydroxyl radical are oxygen-containing free radicals nowknown to be generated in vivo under a variety of normal and pathologicalconditions. An immense amount of work has been done in the last twodecades documenting the deleterious behavior of oxygen radicals. Theseradicals have been implicated as causative agents for everything fromsunburn to aging and have been shown to effect skin and other tissues bydestroying lipid membranes, breaking down DNA, inactivating enzymes, andthe like. As a result of this damage, certain anatomical changes occur,including thinning of the epidermis, thickening of the stratum corneum,reduction of blood supply to the skin, loss of collagen, and formationof age spots, lines and wrinkles.

L-ascorbic acid (vitamin C), a water-soluble antioxidant, can protectfatty tissues from oxy-radical damage by reacting with both superoxideand hydroxyl radicals. It also plays an integral role in collagensynthesis and wound healing by acting as a co-factor for hydroxylationof the proline and lysine residues of procollagen and promotingformation of the triple-helical conformation of mature collagen fibers.This conformation is required for the processing of procollagen tocollagen (D. J. Prockop et al., “Intracellular steps in the biosynthesisof collagen” In: Biochemistry of Collagen, G. N. Ramachandran and A. H.Reddi (Eds.), Plenum Press, New York, 1976, 163-273, C. I. Levene and C.J. Bates, Ann. NY Acad. Sci. 258 [Suppl.]:288-306, 1975). L-ascorbicacid has also been shown to increase both the rate of transcription ofprocollagen genes and stability of procollagen mRNA (S. Tajima and S. R.Pinnell, Biochem. Biophys. Res. Commun. 106:632-637, 1982; B. L. Lyonsand R. I. Schwarz, Nucleic. Acids Res. 12:2569-2579, 1984) as well as tomodulate growth properties of cells (R. Hata et al., Eur. J. Biochem.173:261-267, 1988).

In spite of these important activities of L-ascorbic acid for treatmentof aging, environmental damage, wound healing, and the like, a drawbackof its topical application is its instability. L-Ascorbic acid ischemically defined as an alpha-keto-lactone wherein the number 2 and 3carbons are double-bonded and contain an acid-ionizable hydrogen inwater (pK=4.2). Ascorbic acid is also a moderately strong reductant.These properties, which lead to instability in the ascorbic acidstructure, are well known and have been burdensome to pharmacologistswhen attempting to formulate active ascorbic acid solutions. Forexample, at higher pH, ascorbic acid increasingly is transformed to thenotoriously unstable ascorbate anion. This instability may be due toseveral causes, among which are the following:

a) Stereochemical strain due to polar repulsive forces. As a result,when the 2-hydroxy group ionizes, two negative charges form in closeproximity, thereby favoring ring disruption.

b) Oxidative degradation due to the propensity of the ascorbate anion toact as a reductant. The one-electron oxidation product (dehydroascorbatefree radical) tends to disproportionate, forming another ascorbatemolecule and the two-electron oxidation product (dehydroascorbate),which is extremely unstable in aqueous solution and breaks down toultimately form species such as L-threonic acid and oxalic acid.Transition metal ions can catalyze these reactions.

c) Degradation due to water attack. At lower ascorbic concentrations orionic strength, water itself can react with and degrade the ascorbatemolecule.

For these reasons, among others, scientists working in the field havehad difficulty in formulating stable solutions of ascorbic acid whichwould be useful for cosmetic or dermatological needs. Nevertheless,because of the many beneficial pharmaceutical effects attributed toascorbic acid, numerous attempts have been made to overcome thesedifficulties, as well as user compliance with the extended applicationschedule required, by adding minerals or metabolites and L-ascorbic acidderivatives into the formulation. Several commercial products arecurrently used in cosmetology such as C-Mate (L-ascorbic acid-2-Pmagnesium salt, neutral pH), Cellex-C™ (serum, pH 2.2), ESTER-C®(topical concentrate, pH 6.7), and products from Intaglio® (pH<3.5) andAGERA® (neutral pH). However, the required duration of therapy isrelatively long (weeks to months) and skin irritation will occur withprolonged application of acidic pH formulations.

The cosmetic and therapeutic utility of topically applied Vitamin C andderivatives thereof, is also limited by the lipid-rich stratum corneum,thin layer of skin that acts as highly resistant lipid barrier topenetration of chemical agents into the skin. In both the pharmaceuticaland cosmetic arenas, significant efforts have been put forth in attemptsto overcome the skin's natural barrier to delivery of functional agentsinto the skin topically or into systemic circulation topically. Recentprogress in skin drug delivery has been summarized in several reviewarticles (M. R. Prausnitz, Crit. Rev. Therap. Drug Carrier Syst.14(4):455-483, 1997; A. K. Banga (Ed.), Electrically AssistedTransdermal and Topical Drug Delivery, Taylor & Francis, Bristol, Pa.,1998; A. K. Banga et al., TIBTECH, 16:408-412, 1998; G. Cevc, Exp. Opin.Invest. Drugs 6(12):1887-1937, 1997). Generally, three primary routesacross the stratum corneum are available for molecular transport: (1)Normal or chemically modified skin allows diffusion of small molecules,usually following a tortuous intercellular path within the lipids of thestratum corneum. (2) Transcellular pathways crossing both the cells andintercellular lipids of the stratum corneum can be created byelectroporation to allow passage of chemical compounds. (3) “Shunt”pathways through the hair follicles and sweat ducts may be utilizedduring iontophoresis (IPH), pressure-mediated delivery, and liposomaltransport.

Electroporation is believed to involve the creation of new transientaqueous pathways (pores) in lipid bilayers by the application of a shortelectric pulse having a duration in the range from μsec to sec (D. C.Chang et al. (Eds.), Guide to Electroporation and Electrofusion,Academic Press, New York, 1992; J. C. Weaver, J. Cell. Biochem.51:426-435, 1993; J. A. Nickoloff (Ed.), Methods in Molecular Biology,Vols. 47, 48, 55, Humana Press, Totowa, N.J., 1995) and to drivemolecules through the pores by electrophoresis (M. R. Prausnitz et al.,Proc. Nat. Acad Sci. 90:10504-10508, 1993; M. R Prausnitz, J. Control.Release 40:321-326, 1996; M. R. Prausnitz et al., J. Control. Release38:205-217, 1996; M. R. Prausnitz, et al., Bio/Technology 20:1205-1209,1995; L. Zhang et al., J. Bioelectrochem. Bioenerg. 42:283-292, 1997.For a general discussion of EPT, see co-pending application Ser. No.08/537,265, filed on Sep. 29, 1995, which is a continuation-in-part ofapplication Ser. No. 08/467,566 filed on Jun. 6, 1995, which is acontinuation-in-part of application Ser. No. 08/042,039 filed on Apr. 1,1993 now abandoned, all of which are incorporated herein by reference.

Electrical studies have shown that short, high-voltage pulses can havedramatic and reversible effects on skin electrical properties. During apulse, skin resistance drops as much as three orders of magnitude withinmicroseconds. This alteration in skin resistance exhibits eithercomplete or partial reversibility within minutes or longer. Atrelatively low voltages (<30 V), this drop of skin resistance can beattributed to electroporation of the appendages (e.g., sweat glands andhair follicles). At higher voltages (>30 V), EP of the lipid-corneocytematrix leads to an additional drop of skin resistance Y. A. Chizmadzhevet al., Biophys. J. 74:843-856, 1998. Microscopic imaging suggests thatup to 0.1% of skin area contributes to transport via transcellular andintercellular pathways (U. Pliquett et al., Biophys. Chem. 58:185-204,1996; and M. R. Prausnitz et al., J. Pharm. Sci. 85:1363-1370, 1996).

Alternatives to topical delivery of L-ascorbic acid or its derivativesfor skin improvement include chemical peels, dermabrasion, laser skinresurfacing, or continued large doses of L-ascorbic acid pills, each ofwhich has a considerable discomfort associated with the treatment. Forexample, only a very small portion of L-ascorbic acid ingestedpenetrates into the skin and continuous large oral doses of L-ascorbicacid can cause gastrointestinal discomfort and diarrhea. Chemical peels,dermabrasion, and laser skin resurfacing generally involve a period ofpainful and unsightly healing of disrupted or burned skin surfacelayers.

Thus, there is a need in the art for new and better methods for enhancedtopical delivery of L-ascorbic acid, derivatives thereof, orformulations containing L-ascorbic acid for skin improvement anddermatological purposes without adherence to an extended regimen andwithout substantial discomfort or skin irritation.

BRIEF DESCRIPTION OF THE INVENTION

The present invention overcomes many of the problems in the art byproviding the discovery that penetration of L-ascorbic acid, or acosmetically/pharmaceutically acceptable salt, ester or reducingderivative thereof, through the stratum corneum can be achieved withoutsubstantial pain or skin irritation by topically applying a compositioncontaining such an active agent in conjunction with applying anelectrical impulse to the region of skin. An invention method includesapplying an electric pulse of a sufficient strength and duration to thea region of skin substantially contemporaneously with a compositioncomprising L-ascorbic acid, or a cosmetically/pharmaceuticallyacceptable salt, ester or reducing derivative thereof, to deliver aneffective amount of the L-ascorbic acid or the derivative through thestratum corneum of the region of skin, thereby improving the conditionof the region of skin without substantial pain or skin irritation. Thisinvention has the potential to reduce the duration of skin rejuvenationas compared with conventional techniques.

In one embodiment, an effective amount of such a composition isintroduced into a region of skin by substantially contemporaneouslyapplying an electric pulse for cosmetically improving degenerative skinconditions in a subject in need thereof, for example by enhancingproduction of collagen in the region of skin and/or reducing therein thelevel of free oxygen radicals, and the like.

The invention methods are additionally advantageous when used incombination with other techniques (e.g., iontophoresis (IPH), vibration,phonophoresis, pharmacotherapeutics (optionally, liposome encapsulated))as the combination can produce an additive or synergistic effect so thatmaximal cosmetic and/or therapeutic effects for improving skinappearance are produced. Among these techniques suitable for use incombination with electroporation as described herein, the one currentlypreferred is iontophoresis, a system for promoting topical absorption ofa drug molecule through a skin barrier by generating an electric fieldbetween an anode and a cathode to cause a positively charged molecule tomove from the anode to the cathode, or to cause a negatively chargedmolecule to move from the cathode to the anode (See Journal ofControlled Release, 18:213-220, 1992; Advanced Drug Delivery Review,9:119, 1992; Pharmaceutical Research 3:318-326, 1986).

In another embodiment the present invention provides methods forelectroporation-enhanced dermatological delivery of L-ascorbic acid to asubject in need thereof The invention dermatological delivery methodcomprises applying at least one electric pulse to the surface of aregion of skin substantially contemporaneously with application theretoof a composition comprising ascorbic acid, or acosmetically/pharmaceutically acceptable salt, ester or reducingderivative thereof, said electric pulse having sufficient strength andduration to topically deliver an effective amount of the L-ascorbic acidor the derivative thereof to the region of skin. The inventiondermatological delivery methods have both cosmetic and therapeuticapplications.

BRIEF DESCRIPTION OF THE FIGURE

FIG. 1 is a photograph of a meander electrode that consists of an arrayof interweaving electrode fingers with alternating polarity. The widthof electrode is 2 mm and the gap is 0.2 mm.

FIG. 2 is a simulation plot of equipotential and electrostatic fieldlines generated by the meander electrode of FIG. 1 at a depth 10 μmabove the stratum corneum upon application of an electric potential of120 volts to the skin surface.

FIGS. 3A and 3B are graphs showing distribution of electric fieldsacross skin layers (from the surface of the stratum corneum to a depthof 2 mm in the dermis) under the conditions described in FIG. 2. FIG. 3Ashows the electric field distribution before breakdown of the stratumcorneum by electroporation, and FIG. 3B shows the electric fielddistribution after breakdown of the stratum corneum. The depth of thestratum corneum is indicated by a pair of narrow black bars on the upperleft corner next to the epidermis.

FIGS. 4A and 4B are graphs showing distribution of electric fields atthe depth of 250 μm below the skin surface under the conditionsdescribed in FIG. 2. The distance across the meander electrodes isrepresented along the X-axis. FIG. 4A shows the distribution of electricfields before breakdown of the stratum corneum by electroporation andFIG. 4B shows the distribution after breakdown of the stratum corneum.

FIGS. 5A through 5C are schematic drawings showing an invention handheldpulser. FIG. 5A is a side view showing an adapter hole for a disposablehead and FIG. 5B is a frontal view. FIG. 5C is a schematic drawingshowing an adapter for fastening the handheld pulser to a stationarysurface, such as a table top.

FIGS. 6A through 6E are schematic drawings showing differentconfigurations of disposable heads for use with the invention handheldpulsers. FIG. 6A shows the top view and FIG. 6B shows a side view of asquare type head with clips to attach the head to the body of thepulser. FIG. 6C shows a top view of a moon type head, FIG. 6D shows atop view of a round type head, and FIG. 6E shows a perspective view of aroller type head.

FIGS. 7A and 7B show a meander electrode for use in the inventionhandheld pulser. FIG. 7A shows a top view of the meander electrode andFIG. 7B shows a schematic drawing of a side view the same meanderelectrode, with the width of an individual electrode indicated by doublearrows.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the present invention, there are provided methods fortreating degenerative skin conditions in a subject in need thereof. Theinvention treatment methods comprise applying at least one electricpulse to the surface of a region of skin substantially contemporaneouslywith application thereto of a composition comprising L-ascorbic acid ora cosmetically/pharmaceutically acceptable salt, ester or reducingderivative thereof, said electric pulse having sufficient strength andduration to deliver an effective amount of the L-ascorbic acid, or aderivative thereof through the stratum corneum of the region of skin,thereby improving the condition of the region of skin withoutsubstantial pain or skin irritation. Generally the concentration ofL-ascorbic acid or the derivative thereof in the L-ascorbicacid-containing composition is in the range from about 1% to 35% byvolume, for example from about 20% to 33% by volume.

The invention cosmetic methods can be practiced upon any part of thebody where a degenerative skin condition (i.e. aging) appears. Inhumans, the most commonly treated areas of the body are face, hand, arm,neck, chest, or leg. The term “degenerative skin conditions” is usedbroadly herein, and refers to such symptoms as flabby, sagging skin aswell as wrinkles, age spots, actinic damage caused by UV radiation, andthe like. Thus, degenerative skin conditions can result from eithernatural causes (such as aging), environmental causes (such as pollutionand UV exposure), or such causes as poor diet. Disease conditions whichinhibit endogenous production of collagen and/or oxy-radical scavengers,such as Vitamin C, or disrupt other natural processes that contribute toa healthy, more roseate, elastic skin can also contribute todegenerative skin conditions as the term is used herein. The term“treating” when used in reference to degenerative skin conditionsfurther includes “preventing” or “inhibiting” formation of any of theabove named symptoms of a degenerative skin condition. The inventiondermatological treatment methods can also be used to promote healing ofskin that has been damaged, either by trauma or by surgery.

As used herein, the term “a cosmetically/pharmaceutically acceptablesalt, ester or reducing derivative of L-ascorbic acid” refers to formsof L-ascorbic acid that have been chemically modified to improve thestability and/or bioavailability of the L-ascorbic acid for topicaladministration, including specifically those modifications intended toreduce oxidation of L-ascorbic acid upon exposure to air and/or light,and those modifications intended to increase the solubility ofL-ascorbic acid in lipids. For example, metal salts of L-ascorbic acid,such as sodium or phosphate salts, have increased stability in aqueoussolutions at pH less than about 6.0. L-ascorbic acid 2-phosphate andsodium ascorbic acid are examples of metal salts of L-ascorbic acid.Modifications that increase the solubility of L-ascorbic acid in lipidsinclude esterification, such as is found in the commercially availableproduct Ester-C® or Agera®.

These and other such modifications are disclosed in, for example,Takashima et al, Am. Perfumer & Cosmetics 86:29, July 1971 (esterifyingthe hydroxyl group to form ascorbic acid-3-phosphate and maintaining analkaline pH), U.S. Pat. No. 2,400,171, which discloses stabilizingascorbic acid by converting it to its calcium or zinc salt andpreferably maintaining the pH at 7 to 7.3); and R. Hata et al.,Seikagaku 58:823, 1986, which discloses a more stable phosphatederivative of L-ascorbic acid, L-ascorbic acid-2-phosphate that has beenshown to act as a cofactor for collagen biosynthesis of cells inculture.

Additional components can also be added to the L-ascorbicacid-containing composition to aid in stabilizing the L-ascorbic acid.For example, Ciminera and Wilcox, J. Am. Pharm. Assoc. Sci. Ed. 35: 363,1946) disclose buffering an aqueous solution with an alkaline sodiumsalt), U.S. Pat. No. 4,367,157 discloses stabilizing an aqueous ascorbicacid solution by adding monothioglycerol and maintaining the pH between4 and 7; U.S. Pat. No. 2,442,461 discloses stabilizing calcium ascorbateby adding an aliphatic thiocarboxylic acid and maintaining the pHbetween 5.2 and 5.6; U.S. Pat. No. 2,585,580 discloses stabilizingascorbic acid with thio-sugars and maintaining the pH between 4.0 and6.5.

The composition used in the invention methods may optionally compriseadditional pharmaceutically-acceptable and cosmetically-acceptable safeactive ingredients sufficiently high in purity and sufficiently low intoxicity to render them suitable for application to the skin of animalsand humans. In addition, non-irritating carrier components as are knownin the art can be used that are suitable for delivering the ascorbicacid to the skin. The components must be capable of being commingledwith the ascorbic acid and the other ingredients in such a manner thatthere is no adverse interaction that would substantially reduce theefficacy of the composition during use. Accordingly, the compositionsused in the invention methods may be formulated in a variety of formssuitable for topical administration, such as liquid (e.g., aqueous)suspensions or solutions, lotions, creams, sprays, sticks, ointments,pastes, and cosmetics.

The target for treatment of degenerative skin conditions or for cosmeticpurposes, common referred to as “skin rejuvenation,” is the epidermis.As used herein, the terms “topical delivery” or “topically introducing,”and grammatical variations thereof, refer to the delivery of acomposition into the skin, through/across the lipid bilayer or stratumcorneum, or a combination thereof. An electric pulse that topicallyintroduces a composition into the skin is believed to overcome theresistance of the skin barrier or alter the permeability of the lipidbilayer by substantial reduction in the electrical resistivity of theskin and/or creation of temporary hydrophilic windows in the lipidbilayer, which is also referred to herein as “breakdown” of the stratumcorneum. This process is illustrated by computer simulation in Example 1herein and in FIGS. 3A-B and FIGS. 4A-B. Thus, in a method of theinvention in which an composition comprising L-ascorbic acid or aderivative thereof according to the invention is “topically ortransdermally delivered” into the skin, the composition is driven intoor through/across the skin. The “topically introduced” composition islikely driven across the stratum corneum into the underlying dermallayers, or into the blood supplying such tissue, by the electricpulse(s), to treat the skin tissue.

As used herein, the term “substantially contemporaneously” means thatthe electric pulse and the composition are applied to the region of skinto be treated reasonably close together in time. Preferably, thecomposition is administered prior to or concurrently withelectropulsing. When applying multiple electrical impulses, thecomposition can be administered before or after each of the pulses, orat any time between the electrical pulses. When applying iontophoresis,vibration or ultrasound, the composition can be administered before orafter each, and at any time between.

As used herein, the term “subject” refers to any animal. It isenvisioned that the methods for topically delivering a compositioncomprising L-ascorbic acid, or a derivative thereof, described hereincan be performed on any animal. Preferably, the subject is a human.

As used herein, the term “local,” when used in reference to acomposition, refers to its function in a particular region. Thus, anL-ascorbic acid-containing composition topically introduced into theskin is believed to exert its pharmaceutical or cosmetic activityfunction within the skin. Nevertheless, the skilled artisan willrecognize that some topically introduced compositions may have asystemic effect or function, such that after topically introducing thecomposition into the skin, the composition is distributed to other areasof the subject thereby producing or contributing to treatingdegenerative skin conditions and/or promoting wound healing by acting ata site other than the skin. As used herein, the term “systemic,” whenused in reference to a composition, means that the composition functionsoutside the skin. It is specifically contemplated that compositionsadministered locally may function systemically as well.

In accordance with the invention methods an “effective amount” of anL-ascorbic acid-containing composition is delivered to a treated regionof skin “topically,” e.g., through the stratum corneum. An effectiveamount is an amount effective to produce a desired cosmetic and/ortherapeutic effect, such as improving, preventing or inhibiting adegenerative skin condition or promoting skin healing in a subject inneed thereof. In any event, an “effective amount” is generally a greateramount of the L-ascorbic acid or the derivative than will be deliveredby passive absorption or diffusion, but should not be so large as tocause excessive adverse side effects, such as skin irritation, burning,cytotoxicity, or tissue damage. The amount required for cosmetic ortherapeutic treatment will vary from subject to subject, depending onthe type of formulation, the species, age, and general condition of thesubject (physiological and psychological), the severity of the conditionbeing treated (e.g., chronic vs. acute), the L-ascorbic acid-containingcomposition being employed, and the anatomical region of the skin beingtreated.

For example, the cytotoxicity (MTT₅₀, the amount of a substance requiredto kill 50% of the cells in cell culture) of ascorbate to fibroblastsappears to be at least partially dependent on the age of the subject.Tests of human dermal fibroblasts show that MTT₅₀ was 10,000 ppm forfibroblasts from a 26 year old and 2,500 ppm for fibroblasts from a 52year old (at pH 7.5); However, when MAP (at pH 7.5) was tested as theactive agent, MTT₅₀ was 75,000 ppm for fibroblasts derived from a 26year old and higher than 100,000 ppm for a 52 year old (L. Fan et al.,First World Congress for International Academy of Cosmetic Dermatology,Jan. 28-31, 1999, Malta, Italy). MAP exhibited significantly lowercytotoxicity in fibroblasts of all ages compared to L-ascorbic acid.This data suggests caution when delivering very high concentrations ofL-ascorbic acid or derivatives thereof to the epidermal/dermal junction,or deeper, on a sustained basis.

Thus, although it is not possible to specify an exact “effectiveamount,” an appropriate “effective” amount in any individual case may bedetermined by one of ordinary skill in the art using the teachingsherein. For example, using visual inspection to determine reduction inthe number and/or depth of wrinkles, the amount and/or prominence of agespots, improvement in skin color (i.e., more roseate coloring) oroverall improvement in the condition of skin, or by measuring certainskin parameters, the deterioration of which is associated withdegenerative skin conditions, in response to various amounts of thecomposition, an effective amount can be readily determined. Such skinparameters may include an increase in skin elasticity, an increase inblood supply, a reduced level of free oxygen radicals or enhancedcollagen production in the treated skin region as compared with acomparable untreated skin region. The amount can be adjusted by theindividual or, in the event of any complication, by the physician.

The invention treatment method optionally further comprises furthercomprises additional steps that enhance the permeability of the stratumcorneum of the skin, such as application of a permeation enhancers,microdermalabrasion, and the like. A “permeation enhancer” also can beincluded with electropulsing to increase topical introduction of acomposition into the skin. As used herein, the term “permeationenhancer” refers to any action (e.g., mechanical, physical, chemical) orany composition that can increase or “augment” topically introducing acomposition into the skin. The term “augment,” when used herein as amodifier of topical introduction, means that the rate (over time) oramount of composition topically introduced into the skin viaelectropulsing is greater than that produced by electropulsing in theabsence of the permeation enhancer. Thus, administering a permeationenhancer prior to, substantially contemporaneously with or afterapplying the L-ascorbic acid-containing composition to the skin may“augment” electrically induced topical introduction of the compositioninto the skin. Alternatively, a permeation enhancer can be mixed withthe L-ascorbic acid-containing composition in the pharmaceuticalformulation to be topically introduced. Permeation enhancer compositionsthat increase skin permeability include, for example, alcohols (e.g.,methanol), alkyl methyl sulfoxides (e.g., DMSO), pyrrohidones (e.g.,2-pyrrolidone), surfactants, urea, glycerol monolaurate, polyethyleneglycol monolaurate, glycerol monolaurate, docainehydrochloride,hydrocortisone, menthol, methyl salicylate, and the like. Permeationenhancers further include mechanical or physical actions that functionin association with an electrical impulse (i.e., generally requireapplying an electrical pulse to augment topical introduction of theL-ascorbic acid-containing composition into the skin; e.g., vibration).

In another embodiment according to the present invention, there areprovided methods for electroporation-enhanced dermatological delivery ofL-ascorbic acid to a subject in need thereof. The inventiondermatological delivery method comprises applying at least one electricpulse to the surface of a region of skin substantially contemporaneouslywith application thereto of a composition comprising L-ascorbic acid, acosmetically/pharmaceutically acceptable salt, ester or reducingderivative thereof with the electric pulse having sufficient strengthand duration to topically deliver an effective amount of the L-ascorbicacid or the derivative thereof to the region of skin. The inventiondermatological delivery methods can be modified as described herein withreference to the various embodiments of the invention methods fortreating degenerative skin conditions.

In another embodiment, the invention method further optionally comprisesapplication of pressure to the skin surface after the L-ascorbicacid-containing composition has been topically applied to force thecomposition into pores and hair follicles in the skin. Pressure may beapplied via the electrode during the treatment (to improve contactbetween the skin and electrode) or directly on the skin surface, therebyincreasing topical introduction of a composition into the skin.Preferably, the pressure is applied in conjunction with a movement, suchas a rubbing or stroking of the skin surface, for example, in a “backand forth” or circular motion. However, any convenient means forapplying pressure to the skin surface can also be used, such as manualpressure via a cotton swab or gauze pad.

In another embodiment, the invention method optionally further comprisesapplying iontophoresis (IPH) in combination with an electrical impulseto topically introduce a greater amount of the composition into the skinthan by pulsing alone, or that can drive the composition deeper into theskin, if desired, than by pulsing alone. A switching unit, such as anautomated switch, optionally programmable, could be used to control thetime between applying the impulse and applying IPH, as well asoptionally controlling the time during which IPH is applied. Eachparameter will be determined by the composition introduced, the desiredeffect, the concentration etc. The operation parameters can be set orprogrammed into the mini-generator.

In another embodiment, the invention method optionally further comprisesapplying vibration to the skin surface in combination with an electricalimpulse to topically introduce a composition into the skin. For example,a phonophoresis unit can be used to aid in topically delivering acomposition into the skin by means of ultrasound vibrations. Thus, byapplying vibration or ultrasound before, after or during pulsing and/oriontophoresis on the region of skin surface being treated, thecomposition can be driven deeper into the skin or a greater amount ofthe composition can be driven into the skin than by pulsing alone. Asabove, a switching unit, such as an automated switch, optionallyprogrammable, could be used to control the time between applying theimpulse and applying vibration or ultrasound, as well as optionallycontrolling the time during which impulse, vibration or ultrasound isapplied.

As used herein, the terms “impulse,” “pulse,” “electrical impulse,”“electrical pulse,” “electric pulse,” “electropulse” and grammaticalvariations thereof are interchangeable and all refer to an electricalstimulus. Although the various terms are frequently used herein in thesingular, the singular forms of the terms include multiple pulses.Preferred electrical impulses are pulsed electric fields applied viaelectroporation. The pulse can be unipolar, bipolar, exponential orsquare wave form.

The electric pulse can be provided by any electronic device thatprovides an appropriate electric pulse or electric source sufficient fortopically introducing a composition into the skin. Suitable electricpulses for topically introducing compositions into the skin thereforeinclude, for example, square wave pulses, exponential waves, unipolaroscillating wave forms, bipolar oscillating wave forms, other wave formsgenerating electric fields, or a combination of any of these forms. Eachpulse wave form has particular advantages. For example, square wave formpulses provide increased efficiencies in transporting compounds into thecells in comparison to exponential decay wave form pulses, and the easeof optimization over a broad range of voltages, for example (Saunders,“Guide to Electroporation and Electrofusion,” 1991, pp. 227-47).Preferably, the waveform used is an exponential or a square wave pulse.

An exemplary electric impulse for topically introducing a compositioninto the skin is a pulsed electric field, such as that provided by anelectroporation apparatus. Exemplary pulse generators capable ofgenerating a pulsed electric field include, for example, the ECM600,which can generate an exponential wave form, and theElectroSquarePorator (T820), which can generate a square wave form, bothof which are available from BTX, a division of Genetronics, Inc. (SanDiego, Calif.). Additional electroporation type apparatus arecommercially available and can be used to generate the pulse used in theinvention methods. A pulse generator, physically connected to theelectrode (as well as operationally connected) is preferably portable orlightweight and can optionally be powered by a portable DC power source,such as batteries, optionally being rechargeable.

A typical electrical apparatus for use in practice of the inventionmethods will comprise a mini pulse generator in electrical connectionwith an electrode having a surface, such as a coating of plastic,suitable for application directly to skin. For electroporation throughskin, the goal is an even distribution of an efficacious field. Fortopical delivery of active agents through skin, it is desirable tocontain the electric field to a shallow skin surface layer so that theunderlying nerves and muscles are not subjected to a strong electricalstimulation. After breakdown of the stratum corneum by electroporation,the depth of the electric field is related to the electrode spacing. Anarrow spacing of multiple electrodes will confine the field to asurface region and, therefore is a preferred configuration. For example,depending on the formulation of the composition to be topicallyintroduced, the electrode can be an insulated or porous meanderelectrode, which comprises an interweaving array of metal fingers coatedon a thin film, such as plastic, for placement on skin. A typicalmeander electrode width is in the range of about 0.2 up to about 1 mm,and an electrode gap of about 0.2 mm, wherein the gap can be filled withan electrically insulating substance), as shown in FIG. 1.

However, the nature of the electrode that can be used in the inventionmethod(s) can be varied so long as it is capable of delivering asufficient electric pulse as set forth herein. Thus, a variety ofelectrode types and configurations suitable for practice of theinvention methods. For example, the electrode can be a wire electrode(useful for in vitro studies, and the like). Alternatively, theelectrode can be a plurality of electrodes (e.g., a micropatch electrodeas described in U.S. patent application Ser. No. 09/134,245, filed onAug. 14, 1998, which is hereby incorporated herein in its entirety byreference). Alternatively, the electrode used in practice of theinvention methods can be a porous electrode. The various electrodes usedherein are preferably insulated to protect against excess heat orburning, current leakage, shock, etc. Appropriate electric pulsingparameters are set forth herein or can be determined using the teachingsherein and, in view of these parameters, the skilled artisan can selectamong various suitable electrode types (e.g., ceramic, metal, etc.) andconfigurations (single wire, multiple wire, etc.) available in the art.

Generally, the pulse strength applied to the skin will range from about25 to about 200 volts, preferably from about 25 to about 120 volts andmore preferably from about 50 to about 80 volts. The pulse durationgenerally will be from about 10 microseconds (μsec) to 100 milliseconds(msec), for example, from about 500 μs to about 50 msec, or from about2.0 ms to about 20 ms. There can be one or multiple pulses per cycleapplied at spaced time intervals of about 0.1 msec to about 15 sec.Optionally, the number of pulses is from about 1 to about 30 pulses, forexample, from about 1 to about 15 pulses per train. Generally, a trainof about 5 to 15 pulse cycles are applied, and more than one such trainof pulses can also be used.

Prior to use, the electrode is positioned so that contacts located onthe outer surface are made with the mini generator, and the innersurface of the electrode is positioned to be in contact with the regionof skin to be treated. Substantially contemporaneously, a desiredcomposition is applied topically, to the region of skin to be treated,generally immediately before application of the electrode or while theelectrode is being moved from one region to another on the skin surface.One or more appropriate electric pulses are then applied, preferably asa pulsed electric field.

A means for administering a composition can optionally be used inconjunction with performance of the invention method(s) to administerthe composition to the region of skin prior to, substantiallycontemporaneously with, or after applying an electric pulse,iontophoresis, vibration or ultrasound. Depending on the specificformulation, a composition can be incorporated into a patch reservoir(e.g., similar to a nicotine patch), which is then attached both to theelectrode and the skin surface. Formulations employed for IPH areadvantageously used in this manner.

For human application, efficacy and sensation are the importantcriteria. The results of studies described herein in the Examplesillustrate that there are at least four factors that influence theseimportant considerations: formulation and its corresponding pH value,electrode design, electrical parameters, and skin site. Forcosmeceuticals the preferred formulation is both stable and has aneutral pH. The stability of the formulation determines the period oftime over which the L-ascorbic acid or derivative thereof will be activein the skin to accelerate procollagen processing to collagen anddeposition of collagen in the cell layer. From the standpoint of patientcomfort, the less irritation the product causes, the more acceptable itis. The studies described herein show that topical delivery of neutralor slightly basic compositions is enhanced to a greater degree than thatof more acidic compositions (e.g., pH values <3.0), which are have ahigher rate of passive diffusion. Therefore, electroporation asdisclosed herein is particularly useful for enhancing topical deliveryof L-ascorbic acid-containing compositions at pH values in the lessirritating range from about 4.0-5.0.

In addition, the voltage, waveform type, pulse duration, capacitance,field strength and the number and timing of pulses applied withoutsubstantial pain or skin irritation will vary depending on the locationof the region of skin treated and the formulation of the composition tobe topically introduced. Tests conducted in a live human volunteer,described herein in Example 3, have shown that the face is moresensitive than the forearm, requiring use of a lower voltage and shorterpulses than for the forearm. In addition, the formulation of theL-ascorbic acid-containing composition should also be taken into accountwhen determining the proper pulse parameters to be used to avoidsubstantial pain and/or skin irritation. For example, in these tests(Example 3) the tolerance limit for application of a single electricpulse to the human face was about 80 volts and a pulse duration of about20 msec when the L-ascorbic acid-containing composition applied to theskin is formulated as a cream. By contrast, the level of tolerance forthe human face drops to an electric pulse having a voltage of no morethan about 50 volts and a duration of no more than about 2 msec when theL-ascorbic acid-containing composition is formulated as an aqueoussuspension or solution. For the forearm, relatively higher voltages andlonger pulses are tolerable. For example, testing of the human volunteershowed that the level of tolerance for application of a single pulse tothe forearm was at about 70 volts for a pulse duration of about 10 msecwhen the L-ascorbic acid-containing composition formulated as an aqueoussuspension.

Particular electrical parameters for topically introducing a compositioninto the skin, other than those exemplified herein, can be empiricallydetermined if necessary, in view of the teachings herein and of thegeneral knowledge of those having skill in the art, for example,relating to the electroporation of mammalian cells in vivo.

In accordance with another embodiment of the present invention there isprovided a handheld pulser for applying electric pulses of sufficienttime and duration to cause on or more of electroporation, iontophoresisor electroincorporation. In one aspect of the invention, the pulsercomprises a support member, and an electrode having an optionalelectrically conductive cover (See FIG. 6B), wherein the support memberis of a size and shape to be handheld, and wherein the electrode isattached to the support member and is operatively connected to a pulsegenerator. The pulser may optionally have a controlling means forswitching the pulse generator on and off.

As used herein, “support member” means a rigid body of the kindassociated with, for example, a handheld electric shaver, or the like.Accordingly, the support member may be constructed of any suitablematerial such as plastic, or the like.

As used herein, “electrode” means any electrode that can be adapted foruse in the pulser so long as it is capable of delivering a sufficientelectric pulse as set forth herein. Thus, a variety of electrode typesand configurations are contemplated in the invention apparatus. Forexample, the electrode can be an insulated or porous meander electrode,having an interweaving array of metal fingers coated on a thin film,such as plastic, which can be placed on skin, as is illustrated hereinin FIGS. 7A-B. In another embodiment, the electrode is a plurality ofelectrodes (e.g., a micropatch electrode as described in U.S. patentapplication Ser. No. 09/134,245, filed on Aug. 14, 1998, which is herebyincorporated herein in its entirety by reference). The variouselectrodes used herein are preferably insulated to protect againstexcess heat or burning, current leakage, shock, etc. Appropriateelectric pulsing parameters are set forth herein or can be determinedusing the teachings herein and, in view of these parameters, the skilledartisan can select among various suitable electrode types (e.g.,ceramic, metal, etc.) and configurations (single wire, multiple wire,etc.) available in the art.

As shown in FIGS. 5A-B, it is presently contemplated that the electrodeportion 2 of the pulser 4 be attached to one end of the support member6. Again, using the standard electric shaver as an analogy, theelectrode would be similarly located as the blades of the shaver. Ofcourse any convenient location of the electrode can be employed in thepractice of the present invention.

Invention pulsers may be used by lay people or professionals.Accordingly, it is contemplated that invention pulsers can have useradjustable options of varying degrees of sophistication. Accordingly, asshown in FIG. 5B, the invention apparatus can have a variety offunctionalities in addition to the optional controlling means 8 (“switchbutton”) for applying an electric pulse. For example, the apparatus canhave an indicating means 10 and 12, which can be selected to indicatesuch parameters as “apparatus ready,” the various pulse parametersettings (e.g., voltage, capacitance, pulse duration, time delay betweenpulses, pulse wave type), pulse(s) applied, parameters of the appliedpulse(s) (e.g., voltage, capacitance, pulse duration, pulse wave type,number of pulses) or a combination thereof. In addition to visibledisplays as shown in FIG. 5B, indicating means can be audible, or acombination of visible and audible. For example, a single audible “beep”can indicate that the “apparatus is ready,” two audible “beeps” canindicate that a pulse has been correctly applied and three audible“beeps” can indicate a malfunction or that the pulse was not or wasimproperly applied. Visual indicating means include analog or digitalalpha-numeric displays (e.g., LCD, LED and the like), as in watches, andfurther can include illuminating means for low light visualization, forexample, by white light, electroluminescent backlighting for LCD orelectroluminescent lamps (i.e. INDIGLO™), or by various fluorescent orradioactive illuminating compositions, and the like.

As shown in FIG. 5C, the invention pulser may optionally be fitted withan adapter 22 for fastening the handheld pulser to a stationary surface,such as a table top.

Additional “user friendly” functions include the aforementionedcontrolling means for applying an electric pulse (e.g., pushbutton,knob, lever switch, dial and the like) as well as means for adjustingparameters (e.g., pushbutton, knob, lever switch, dial and the like)including, for example, pulse duration, voltage, capacitance, fieldstrength, number, and wave type. Means for adjusting, setting, storingor retrieving one or more pulse parameters also are included herein.Such means include traditional mechanical electronic controls (e.g., aselector switch controlling each parameter in which the switch has aplurality of settings; as well as a chip control (e.g., silicon wafertypes commonly used in the computer industry) which is controlled, forexample, by a pushbutton interface, as in watches for example. A chip,optionally removable from the apparatus or, user and/or manufacturerprogrammable for control of the various pulse parameters set forthherein also is contemplated. Storage capacity of such a chip issufficient to provide virtually unlimited fine control of the variousparameters, as well as storing different pulse parameter settings fordifferent compositions, users and the like. As each of the variouselectronic functionalities of the invention apparatus described hereincan be controlled or managed by a computer chip, a chip affords theoption of additionally incorporating software, if desired, said softwareoptionally user programmable.

Depending on the formulation of the therapeutic agent to be introducedinto the skin and the condition of the skin, it may be advantageous toapply one or more additional treatment modalities to facilitate theintroduction of the therapeutic agent. Such additional modalities mayinclude vibration, phonophoresis, and the like. Accordingly in anotheraspect of the present invention, a vibration unit also can optionally beincluded in the apparatus, which can be used in combination with anelectrical impulse to introduce a composition into the skin. Aphonophoresis unit, which can transdermally introduce a composition intothe skin by means of ultrasound, also can optionally be included in theapparatus, if desired. Thus) by applying vibration or ultrasound before,after or during pulsing and/or iontophoresis on the skin, thecomposition can be driven deeper into the skin or a greater amount ofthe composition can be driven into the skin than by pulsing alone. Asabove, a switching unit, such as an automated switch, optionallyprogrammable, could be used to control the time between applying theimpulse and applying vibration or ultrasound, as well as optionallycontrolling the time during which impulse, vibration or ultrasound isapplied.

A means for administering a composition can optionally be included inthe electrical apparatus, which can be used to administer thecomposition to the skin prior to, substantially contemporaneously with,or after applying an electric pulse, iontophoresis, vibration orultrasound, in their various embodiments. Depending on the specificformulation, a composition can be incorporated into a patch reservoir(e.g., as a nicotine patch), which is then attached both to theelectrode and the skin.

Invention apparatus is adaptable to a number of topical applicationneeds, and is similarly adaptable to a number of different bodysurfaces. Accordingly, in one embodiment of the present invention, theelectrode is detachable, thereby allowing different electrode typesand/or shapes to be employed. In a particular aspect of the presentinvention, the electrode can be attached to the support unit by avariety of suitable means known to those of skill in the art. Forexample, the electrode can be directly attachable to the support meansby a mechanical attachment (e.g., clips, or the like). In another aspectof the present invention, the electrode may be attached to a mountingbracket which is, in turn, attachable to the support unit. Electrodesmay be mounted to the detachable mounting bracket by means of anintegral adhesive means (such as an adhesive strip, as shown for examplein FIG. 6B, or the like).

As used herein, “mounting bracket” means a device fabricated from anysuitable material for mounting an electrode thereto. Depending on howcurrent is to be conveyed from the pulse generator to the electrode,suitable materials can be non-conductive (e.g., plastics, polymerresins, or the like), or conductive (e.g., metal, metal alloy, or thelike).

Mounting brackets, like electrodes can have a variety of shapes toaccommodate the different body surfaces encountered in use. Accordingly,as depicted in FIGS. 6A-E, the electrode or mounting bracket can besquare, round, crescent (or moon-shaped), tubular, or the like. The tubeshape is particularly useful when employed in conjunction with an axlerunning through its longitudinal axis, as shown in FIG. 6E. In thismanner, the electrode can be rolled along the skin surface rather thanbeing slid along the skin surface as with a static electrode. Contactregements located along the side of the roller will dispense thetreatment fluids while the roller turns. This may be of particularadvantage when treating delicate skin or when abrasion is otherwise aconcern. Of course, there are means known to those of skill in the artfor reducing the friction between a static (i.e., non-rolling) electrodeand the skin. Reduced friction surfaces may be employed so long a thesurface material allows pulse transmission to the skin.

Alternatively, as shown in FIG. 6B, an electrode cover 14, for examplemade of an elastic material, and a cosmetic reservoir 16 can be placedatop the electrode 18, and the electrode 18 can be backed with anadhesive film 20 that can be peeled off for use.

The optional electrode cover can be manufactured of essentially anymaterial compatible with applying an electrical impulse to the skin. Theelectrode cover can be made of a single material type or can be made ofmultiple material types. In one embodiment, the electrode cover ismanufactured of a single flexible cushioned or compressible material,e.g., elastic-containing cotton, or the like, as shown, for example inFIG. 5. Preferably, the various electrode cover embodiments of theinvention apparatus are hypo-allergenic, non-allergenic or so modifiedto be non-allergenic.

It may be desirable to couple a dispensing unit to the handheld pulser.In this aspect of the present invention, the pulser has a unit attachedthereto for containing and dispensing the agent to be applied to theskin The dispensing unit may optionally be controllable to dispense ameasured amount of agent. The dispensing unit can be any devicecompatible with the electronic pulse to be delivered by the pulser, andis contemplated to include passive devices such as, for example, areservoir-type patch or sponge; and active devices such as a pump oreven an injection means, such as a syringe, or the like.

In addition to efficacy, both sensation and user safety are important.Thus, in another embodiment, the invention further provides an apparatushaving means for preventing applying excess pulse voltage, duration,field strength and/or number. Any means which passively or activelyinterrupts or disrupts the electric circuit, including fuses, circuitbreaker switches, and the like, or devices that actively monitor thevarious pulse parameters and interrupt or disrupt the electric circuitto prevent excess pulse voltage, duration, field strength, pulse numberfrom being applied can be incorporated into the circuit path. Thoseskilled in the art of electrical devices will know of other protectiveelements that prevent applying excess pulse voltage, duration, fieldstrength or number.

The electric pulse can be provided by any electronic device thatprovides an appropriate electric pulse or electric source sufficient forintroducing a topically applied composition into the skin. Suitableelectric pulses for transdermally topically applied composition into theskin therefore include, for example, square wave pulses, exponentialwaves, unipolar oscillating wave forms, bipolar oscillating wave forms,other wave forms generating electric fields, or a combination of any ofthese forms. Each pulse wave form has particular advantages; square waveform pulses provide increased efficiencies in transporting compoundsinto the cells in comparison to exponential decay wave form pulses, andthe ease of optimization over a broad range of voltages, for example(Saunders, “Guide to Electroporation and Electrofusion,” 1991, pp.227-47). Preferably, the waveform used is an exponential or a squarewave, or bipolar oscillating wave forms.

An exemplary electric impulse for introducing a topically appliedcomposition into the skin is a pulsed electric field, such as thatprovided by an electroporation apparatus. Because the apparatus of thepresent invention is designed to be handheld, it is presently preferredthat the pulse generator be integrated into the support member. Power tothe pulse generator can be supplied by any suitable means, includingdisposable battery, rechargeable battery, household AC current, or thelike. Alternatively, the pulse generator may be a “table top” unit,wherein the pulse is transmitted to the handheld pulser via conductingwire, or the like. Exemplary pulse generators capable of generating apulsed electric field include, for example, the ECM600, which cangenerate an exponential wave form, and the ElectroSquarePorator (T820),which can generate a square wave form, both of which are available fromBTX, a division of Genetronics, Inc. (San Diego, Calif.). Additionalelectroporation type apparatus are commercially available and can beused to generate the pulse for the invention apparatus and in practicingthe invention methods. Such pulse generators can be operativelyconnected to the pulse applicator, or alternatively can be physicallycontained within the pulse applicator. A pulse generator, physicallyconnected, is preferably portable or lightweight, and an optionalportable DC power source, such as batteries, optionally beingrechargeable, can be included to provide the power source to the pulsegenerator.

The following examples are intended to illustrate but not limit theinvention. While they are typical of those that might be used, otherprocedures and applications of the invention methods known to thoseskilled in the art may alternatively be used.

EXAMPLE 1

Computer Simulation

For computer simulations, the skin structure was reduced to abiophysical model wherein a very thin (typically 15-30 μm), highlyresisitive layer, representing the SC, covers a thicker, highlyconductive layer, representing the epidermis and dermis. Field plots(V/cm) were used in order to get a better understanding of fieldsgenerated by meander type electrodes in human skin The objective was tosolve Laplace's equation for electrostatic potential, (∇²V=0, and thenfind a solution for field strength from the equation E=−∇V throughoutthe electrostatic environment. EMP software (Field Precision, N. Mex.)was used for this purpose, and data was obtained to import into theplotting programs, as follows.

The simulation model contained a meander electrode comprised of sixindividual electrodes of alternating polarity, each electrode being 1 mmwide, with a 0.2 mm space between adjacent electrodes, as illustrated inFIG. 1. The simulation further included immersion of the electrodes in asaline solution that had a resistivity of 1 kohm-cm and placement of theelectrode approximately 10 μm above the stratum corneum. In the firstrun of simulation, the stratum corneum was selected to have a thicknessof 15 μm with a resistivity of 6.0×10⁵ kohm-cm. The underlying epidermisand dermis was selected to have a thickness of 2 mm with a resistivityof 5 kohm-cm. Specific surface resistivities were calculated by themodel and these numbers were divided by their respective skin layerthicknesses. The potential applied was 120 V. A graph showing thesimulated field strength below a single electrode in the meanderelectrode with tissue depth (from zero to 0.2 cm) before breakdown ofthe stratum corneum is shown in FIG. 3A.

A second run of simulation was performed as in the first run, exceptthat the resistivity of the stratum corneum was made equivalent to theresistivity of the epidermis to simulate the breakdown of the stratumcorneum as would happen if transient aqueous pathways or pores in thelipid bilayers were created. The potential applied was again 120 V.Finally, the distribution of electric fields at different depths of theskin was calculated using the model for a time t=0, before the breakdownof the SC, and also for a time t>0 after the breakdown of the stratumcorneum. FIG. 3B is a graph showing the simulated field strength below asingle electrode in the meander electrode at a range of tissue depthfrom zero to 0.2 cm with skin resistivity reduced to simulate thebreakdown of the stratum corneum.

The results of these calculations show that before breakdown of thestratum corneum (FIG. 3A), the field strength in the stratum corneum wasfive orders of magnitude higher than in the epidermis (<0.2 V cm⁻¹).After breakdown of the stratum corneum, the field strength increased byfour orders of magnitude within the epidermis region at a skin depth of125 μm (FIG. 3B). Interestingly enough, the increase of field strengthalways occurred around the edge of the electrodes (FIGS. 4A and 4B). Thearea below the center of each electrode had the lowest field.

The field strength data was also obtained from the simulation modelacross the skin surface for a width (0.26 cm to ≦0.5 cm) greater thanthe width of two electrodes in the meander electrode to determine therelationship between distance from the electrode and the field strengthgenerated in skin at a constant depth of 250 μm. The results of thesecalculations, shown in FIG. 2, indicate that the potential drop betweenthe electrodes in the meander electrode is mainly confined to thestratum corneum.

EXAMPLE 2

In Vitro Electroporation-assisted Delivery of L-Ascorbic Acid

In vitro delivery studies were conducted to determine the relativeefficacy of two L-ascorbic acid formulations, one in cream formulationand one having crystals of L-ascorbic acid in solution. For the creamformulation, Intaglio® cream((pH 3.5, Research Institute for Plastic,Cosmetic and Reconstructive Surgery, Inc., San Diego) was used, whichcontains 20% L-ascorbic acid. This cream formulation has been used inphysicians' offices for skin resurfacing. For the aqueous suspensionformulation, 2 g of L-ascorbic acid crystals were suspended in a vialcontaining 6 ml of distilled water at room temperature (pH 1.86). Thevial containing the suspension was wrapped with aluminum paper and waskept in a cold water bath during experiments to prevent oxidization ofL-ascorbic acid. Under light microscopy, the crystals had the shape ofneedles, or small diamonds (approx. 10-50 μm in length).

The in vitro tests were conducted using two types of human skin. A fullthickness of human forearm cadaver skin (52-year-old man) obtained fromThe National Disease Research Interchange (Philadelphia, Pa.) and freshhuman skin received immediately after plastic surgery (The ResearchInstitute for Plastic, Cosmetic and Reconstructive Surgery, Inc.) Thefresh skin samples were from the neck and face of both males andfemales. Full thickness skin was prepared for use in the experiments bytrimming away the fat under the dermis. The thickness was 1.2 mm forfemale neck skin and 1.8 mm for skin from other locations.

Experimental Setup

In order to control the environment for human cosmetic applications, acustom-made glove box was constructed for the study of clear plasticwith dimensions of 60 cm×40 cm×30 cm. The glove box was fitted with alight bulb, an electrical fan, a heater, a humidity controller (set at40% humidity), and a thermostat (set at 35°±1° C.). The diameter of theglove hold was about 21 cm. The top of the box could be removed totransport experimental items, but the box was sealed during operation.Temperature was determined to be steady prior to the experiments. Twooutputs from a pulse generator were connected to electrodes through awall opening in the box.

An exponential pulse generator (Model ECM600, BTX, a Division ofGenetronics Inc., San Diego, Calif.) connected to meander electrodes(Model P/N 454-P, Genetronics Inc.) were used for all experiments. Thearea of electrode exposed to the skin during pulsing was 1-1.5 cm²,varying with the size of skin samples. The skin was placed on a brassmetal plate during the pulsing, with the meander electrode located ontop of the stratum corneum. The metal plate was used as a substitute forconductive underlying tissue for the in vitro study. Skin resistance wasmeasured prior to the experimental process. It was 100-150 kohms and300-800 kohms for cadaver and fresh surgical skin, respectively. Theprotocol for electroporation of the cadaver skin was as follows: 100 Vfor 20 msec, 6 pulses with 15 sec time intervals between pulses. Forfresh skin, the protocol was: 60 V for 30 msec, 6 pulses for cream; 60 Vfor 2.7 msec and 5 msec, 6 pulses for the suspension (with 2.5 sec timeinterval between pulses to shorten the time of the treatment and reducethe loss of L-ascorbic acid).

For a control, the conventional cosmetic procedure for cosmetic skinresurfacing of humans in physicians' offices was simulated. Theconventional procedure involves applying Intaglio® cream on facial skin,placing a finger on the skin surface, and making a few circles clockwiseand counter clockwise. In the simulated control procedure, a force (300g) from a calibrated spring-loaded plastic cylinder was applied to thesurface of the skin samples through the meander electrode. The pressurewas held constant during pulsing at 200 g cm⁻². Then, the cylinder wasmoved gently 6 times clockwise and counter clockwise, maintainingpressure for a total of 30 sec.

A further control studied the natural absorption of L-ascorbic acidsuspension on the skin (with no pressure and massage and no pulsing).

The final step for each of the test and control groups was washing theL-ascorbic acid-containing composition from the surface of the skinsamples quickly and gently. The skin surface was wiped with cottonQ-tips 4 times in the following sequence: wet-dry-wet-dry. Then the skinsample was immediately placed in a dry petri dish with the stratumcorneum on the bottom the skin was cut from dermis to stratum corneuminto two parts, each piece was put in a small centrifuge tube, and lefton dry ice for the HPLC assay to determine the concentration of theL-ascorbic acid that penetrated into each skin sample.

HPLC Analysis

Each frozen sample was weighed and 300 μl of extraction solution (0.1%methylprogesterone acetate (MPA), 0.1 mM EDTA) were added to eachsample. Each sample was homogenized for one minute with a tissuegrinder. The tip of the grinder was rinsed with 500 μl of extractionsolution, and the final volume was brought to 1 ml with extractionsolution. Appropriate sample dilutions were prepared with the extractionsolution.

Electrochemical HPLC was performed to determine the amount of L-ascorbicacid per gram of skin using an ion exclusion column (BioRadLaboratories, Richmond, Calif.) at 30° C. The mobil phase was 0.001 MH₂SO₄ with a flow rate of 0.6 ml min⁻¹. The injection volume was 10 μl.An ESA Coulochem Detector was used with settings at 0.4 V, gain 10X×1.The retention time was 10.0 min. Standard curves were prepared forL-ascorbic acid concentrations of 1, 2, 5, 10, and 30 μg L⁻¹.

The results of these experiments show that for the cream formulation (pH3.5) electroporation enhanced penetration of L-ascorbic acid by 38% (seeTable 1 below); whereas for the suspension formulation (pH 1.86) therewas no significant improvement of L-ascorbic acid penetrationattributable to electroporation. The control group (pressure andmassage, but no pulsing) showed the effect of pushing the L-ascorbicacid-containing composition into the skin.

TABLE 1 Results from human cadaver skin Formulation (% of L-ascorbicacid Asc in the skin (mg g⁻¹ of skin) (Asc) No Pulsing Pulsing Cream(20%) 0.47 ± 0.18 0.65 ± 0.43 Suspension (w/v: 33%) 5.65 ± 3.68 6.03 ±2.24 100 V, 20 ms, 6 pulses; n = 3 − 5

By contrast, the natural absorption of L-ascorbic acid suspension on theskin (with no pressure and massage and no pulsing) was measured afterthe same washing steps (see Table 2). The comparatively high absorptionof the suspension may be due to the lower pH of this composition (pH1.86).

TABLE 2 Results from basic controls by HPLC HPLC test Skin source(background of Asc) (Asc mg g⁻¹ of skin) Cadaver skin 0.01 Freshsurgical skin Female's facial skin 0.01 Male's facial skin 0.02 Passiveabsorption of Asc in the cadaver skin 0.11 (suspension 33%) LabeledVitamin C sample HPLC test Suspension (28% and 33%) 28.5% and 36.8%Cream (20%) 19.7% Sigma Vitamin C (5 μg ml⁻¹) 4.95 μg ml⁻¹

For the fresh surgical skin model, the results ofelectroporation-mediated topical delivery of L-ascorbic acid (Table 3)show that L-ascorbic acid penetration was increased 300% compared to thecontrol with the cream formulation, and there was approximately a 54%increase for the suspension formulation.

TABLE 3 Results from human fresh surgical skin Formulation Asc (mg g⁻¹of skin) % of Asc No pulsing Pulsing Cream 20% 0.075 ± 0.007  30 ms 0.22± 0.12 Suspension 2.600 ± 0.70 2.7 ms 3.05 ± 1.00 (w/v: 33%)   5 ms 4.01± 0.90 60 V, 6 pulses; n = 2 (cream), n = 3 − 5 (suspension)

This result may be attributed, in part, to the lower pH of thesuspension formulation and to the lower test voltage and shorter pulselength used with the suspension formulation, which was selected as aresult of human tolerance information provided from the human volunteertest described herein in Example 3 below.

Surprisingly, the results (Table 3) of the basic control studies showthat passive absorption of L-ascorbic acid in the cadaver skin samplesfor the 33% suspension was an order of magnitude higher than for thefresh surgical skin samples.

EXAMPLE 3

To obtain the tolerability of electrical sensation with reference to thepulsing parameters, such as voltage and pulse length, a pilot humanvolunteer was tested. It was expected that the nerve sensation would berelated to three variables, or a combination thereof. (i) pulseparameters, (ii) the L-ascorbic acid formulation (i.e., whether cream orsuspension) and (iii) location of the skin site. Not surprisingly, thefacial skin was found to be more sensitive than the forearm skin duringthe electroporation. On the facial skin, the tolerance threshold pulseparameters for a single pulse turned out to be 80 V and 20 msec (for thetest cream), and 50 V and 2 msec (for the test suspensions). On theforearm, higher tolerance (for the test suspensions) was observed at 70Vand 10 msec.

While the invention has been described in detail with reference tocertain perferred embodiments thereof, it will be understood thatmodifications and variations are within the spirit and scope of thatwhich is described and claimed.

1. An apparatus for delivering voltage pulses to tissue so as toestablish an electric field sufficient to assist the topicalintroduction of a therapeutic agent into a cell of a tissue, wherein theapparatus comprises: a) a detachable electrode support member havingdisposed thereon one or more opposing pairs of electrodes arrangedrelative to one another to form an electrode array, and furthercomprising an electrode cover and a cosmetic reservoir disposed betweenthe electrode cover and electrode array, wherein the cosmetic reservoiris in fluid communication with the electrode array; and b) a powersupply in electrical communication with the pair(s) of electrodesdisposed in the support member, wherein the power supply providesvoltage pulses to at least a pair of the electrodes to effectelectroporation.
 2. The apparatus according to claim 1, wherein saidapparatus is configured to be hand-held.
 3. The apparatus according toclaim 1, wherein said reservoir comprises a porous reservoir.
 4. Theapparatus according to claim 1, wherein said detachable electrodesupport member is square, round, contoured, or tube shaped.
 5. Theapparatus according to claim 1, wherein said electrodes are a meandertype electrode or a micropatch electrode.
 6. The apparatus according toclaim 5, wherein said meander type electrode comprise an interweavingarray of electrically conductive electrode fingers coated on a thinfilm.
 7. The apparatus according to claim 5, wherein said meanderelectrode is insulated or porous.
 8. The apparatus according to claim 1,wherein said electrodes have a width of about 1 mm.
 9. The apparatusaccording to claim 1, wherein said electrodes are separated by a gap ofabout 0.2 mm.
 10. The apparatus according to claim 1, wherein said pulsegenerator is powered by a battery.
 11. The apparatus according to claim1, further comprising a vibrating unit.
 12. The apparatus according toclaim 11, wherein said vibrating unit applies ultra sound.
 13. Theapparatus according to claim 1, further comprising a phonophoresis unit.14. The apparatus according to claim 1, further comprising a pressuresensor unit.
 15. The apparatus according to claim 1, further comprisinga unit to measure and record the skin resistance of the subject.
 16. Theapparatus according to claim 1, further comprising a means for applyingiontophoresis.
 17. The apparatus according to claim 16, wherein saidmeans further comprises a switching unit to allow for switching betweenelectroporation and iontophoresis.
 18. The apparatus according to claim1, wherein said pulses are electrical impulses.
 19. The apparatusaccording to claim 18, wherein said pulses are selected from the groupconsisting of unipolar, bipolar, exponential and square wave forms. 20.The apparatus according to claim 1, wherein said electrodes are selectedfrom the group consisting of wire, porous, and meander electrodes.